Physiologic cold shock of Moraxella catarrhalisaffects the expression of genes involved in the iron acquisition, serum resistance and immune evasion
© Spaniol et al; licensee BioMed Central Ltd. 2011
Received: 8 April 2011
Accepted: 12 August 2011
Published: 12 August 2011
Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence.
A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis.
Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.
Moraxella catarrhalis colonizes the mucosal surface of the human nasopharynx and is a major cause of acute otitis media in children and of exacerbations of chronic obstructive pulmonary disease in adults [1, 2]. Clinical studies have revealed that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis displays seasonal variation and increases in winter [3–6]. Because breathing cold air (e.g., -1°C at 10-20 l/min) reduces the nasopharyngeal temperature from 34°C at room temperature to ~26°C within several minutes and for extended periods of time , the human nasopharyngeal flora is repeatedly exposed to rapid downshifts of environmental temperature. In addition to viral infections that pave the way for subsequent secondary bacterial infections , the rapid downshift of temperature induces adaptive events in the residential upper respiratory tract flora that may lead to the transition from asymptomatic colonization to bacterial secondary infection. Our previous findings established that a 26°C cold shock upregulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, and promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin [9, 10]. Exposure of M. catarrhalis to 26°C also increases the outer membrane protein (OMP)-mediated release of the proinflammatory cytokine IL-8 in pharyngeal epithelial cells and reduces the expression of porin M35, which may affect the resistance to aminopenicillins [10, 11].
Among the various putative virulence factors that have been identified to date, several other proteinaceous antigens including lactoferrin-binding proteins (LbpA/B), transferrin-binding proteins (TbpA/B), CopB, UspA2 and Hemagglutinin (Hag/MID) may be involved in the cold shock response and thus be important in adapting to and colonizing the human host. Iron is an essential nutrient for most bacteria and M. catarrhalis overcomes the host's restriction of free iron through the evolution of iron acquisition systems which enable it to use lactoferrin, transferrin, hemoglobin, and hemin as iron sources. The primary site of M. catarrhalis entry into the human host is the nasopharynx, where lactoferrin is the predominant source of iron. Therefore, efficient iron acquisition from lactoferrin is an important virulence factor for pathogenic bacteria. The surface protein CopB is involved in the ability of M.catarrhalis to acquire iron from human transferrin and lactoferrin and is also an important factor responsible for complement evasion [12, 13]. UspA2, a major OMP of M. catarrhalis, binds vitronectin, a component of both plasma and the extracellular matrix, and confers serum resistance of M. catarrhalis . Furthermore, the UspA2 is able to bind human C3 and C4bp protecting M. catarrhalis from complement-mediated killing [15, 16]. The surface protein Hag/MID that acts as an adhesin and hemagglutinin, exhibits unique immunoglobulin (Ig) D-binding properties and binds to both soluble and membrane-bound IgD on B cells [17–19]. Our previous study demonstrated that exposure of M. catarrhalis to 26°C down-regulates hag mRNA expression , indicating a possible involvement of Hag in the cold shock response.
In the present study we investigated the effect of a 26°C cold shock on the expression of genes involved in iron acquisition, serum resistance and immune evasion. Cold shock induced the expression of genes involved in transferrin/lactoferrin acquisition and enhanced binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulated the expression of UspA2, a major OMP involved in serum resistance, leading to the improved vitronectin binding. In contrast, cold shock decreased the expression of Hag, a major adhesin mediating B cell response, and reduced IgD-binding on the surface of M. catarrhalis.
Bacterial strains and culture conditions
M. catarrhalis strain O35E, its isogenic tbpB (O35E.tbpB), uspA1 (O35E.uspA1), uspA2 (O35E.uspA2), hag (O35E.hag) and lpxA (O35E.lpxA) mutants, and clinical isolates 300 and 415 have been described elsewhere [9, 20, 21]. Bacteria were cultured at 37°C and 200 rpm in brain heart infusion (BHI) broth (Difco) or on BHI agar plates in an atmosphere containing 5% CO2. Cold shock experiments were performed as described . Bacteria were grown overnight at 37°C, resuspended in fresh medium and grown to mid-logarithmic phase (optical density at 600 nm [OD600] of 0.3). Subsequently, bacteria were exposed to 26°C or 37°C, respectively, for 3 hours (unless otherwise stated). The growth rates of M. catarrhalis under iron depletion conditions were evaluated by culturing the bacteria in BHI containing 30 μM desferioxamine (Desferal; Novartis).
RNA for mRNA expression analysis was isolated and used for complementary DNA (cDNA) synthesis as described elsewhere . Generated cDNA was amplified by semi-quantitative polymerase chain reaction (PCR) using primers for lbpB (5'-GCAAGGCGGTAGGGCAGAT-3', 5'-CCTGCTTTTTCGGCGGTGTC-3'), lbpA (5'-AACAACGCATTCACAGCACCGATT-3', 5'-GATACCAAGACGAGCGGTGATG-3'), tbpB (5'-CAAGCAGGCCGGTGGTATGG-3', 5'-GGTAAATGGGGTGAATGTGGTTGC-3'), tbpA (5'-AAGGCGGAGGCAACAGATAAGACA-3', 5'-AGAGCCAGATAATGCCCCAGAGC-3') and 16S ribosomal RNA [rRNA] (5'-AAGGTTTGATC(AC)TGG(CT)TCAG-3', 5'-CTTTACGCCCA(AG)T(AG)A(AT)TCCG-3').
Quantitative real-time PCR was performed in triplicate for both target and normalizer (16S rRNA) genes. No-template controls and RT-negative controls were included in each run. Primers and probes were purchased from Applied Biosystems. Primers for tbpB were 5'-TCCTTTCACTTCGCTAAATCGGTTT-3', 5'-CCACACAAGATGCGGTCAAATATAAA-3', and TaqMan probe was 5'-(FAM)CCTTTGTTGGCAACATC-3'. Primers for uspA2 were 5'-GCCTTAGACACCAAAGTCAATGC-3', 5'-AAGCTGCCCTAAGTGGTCTATTC-3', and TaqMan probe was 5'-(FAM)TGAAAACGGTATGGCTG-3'. Primers and probes for hag and 16S rRNA were used as described elsewhere [9, 22]. Relative quantification of gene expression was performed using the comparative threshold method. The ratios obtained after normalization were expressed as folds of change compared with samples isolated from bacteria exposed for 1 h at 37°C.
OM vesicles, composed of OMPs and lipooligosaccharide (LOS), from strain O35E exposed for 3 h to either 26°C or 37°C were prepared by the EDTA buffer method . Samples were resolved by SDS-PAGE using a 7.5% polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore). Lactoferrin binding was detected using mouse anti-human lactoferrin monoclonal antibody (AbD Serotec) and horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Sigma). IgA-binding was detected using human saliva samples as the primary antibody source and HRP-conjugated goat anti-human IgA (Sigma) as secondary antibody. Sampling of saliva from healthy volunteers was approved by the local ethics committee.
Solid-phase lactoferrin binding assay
Detection of lactoferrin binding to M. catarrhalis was performed as described elsewhere . Equal amounts of strain O35E grown at 26°C or 37°C for 3 h were spotted onto the nitrocellulose membranes. The blots were blocked in Tris-buffered saline (50 mM Tris buffer containing 0.1 M NaCl [pH 7.0]) containing 0.5% nonfat dry milk and incubated with human lactoferrin (10 μg/mL), followed by a mouse anti-human lactoferrin antibody and developed by using horseradish peroxidase-conjugated goat anti-mouse antibody.
Two-dimensional gel electrophoresis (2-DE)
Treatment of M. catarrhaliswith lactoferrin
Treatment of M. catarrhalis with lactoferrin was performed as described elsewhere . Strain O35E was grown to an OD600 of 0.5, resuspended in assay solution containing 0.1% gelatine to a concentration of 105 CFU/mL prior to the addition of lactoferrin (1 mg/mL, Sigma). Samples were incubated at 37°C for 1 and 3 h followed by plating on BHI agar to determine viability.
Bacteria were exposed to 26°C or 37°C for 3 h. The OD600 was adjusted to 0.2, the 200-μL aliquots were washed in PBS-1% BSA, and incubated with 1 μg/mL of lactoferrin or with 1 μg of vitronectin (Millipore) for 1 h. To assess the ability of M. catarrhalis to bind salivary lactoferrin, bacteria were preincubated with saliva samples (1:20 dilution) from healthy adults. Bacteria were incubated with mouse anti-human lactoferrin monoclonal antibody (AbD Serotec) or mouse anti-human vitronectin monoclonal antibody (Quidel) followed by incubation with Alexa 488-conjugated goat anti-mouse antibody (Invitrogen) and analyzed on a FACScan cytometer using CellQuest software (version 4.2; BD Bioscience). Anti-human lactoferrin or vitronectin antibodies and Alexa 488-conjugated anti-mouse antibody were added separately as negative controls.
Binding of transferrin to M. catarrhalis was analyzed using fluorescein isothiocyanate (FITC)-conjugated human transferrin (0.1 μg/mL, Jackson Immunoresearch).
The ability of M. catarrhalis to bind human IgD was analyzed as described elsewhere . Strain O35E, Hag-deficient mutant (O35E.hag), LOS-deficient mutant (O35E.lpxA) and clinical isolate 300 were exposed to 26°C or 37°C for 3 h, harvested, and incubated with 50% of pooled normal human serum (NHS) as a source of IgD, followed by a FITC-conjugated rabbit anti-human IgD polyclonal antibody (Dako).
The expression of UspA1/A2 and CopB was analyzed using the uspA1/A2-specific 17C7 and the copB-specific 10F3 (1:20) mouse monoclonal antibodies.
Data were expressed as mean ± 1 standard deviation (SD). Differences between groups were analyzed by one-way analysis of variance with a Bonferroni posttest using Prism software (version 5.01; GraphPad). P< 0.05 was defined as statistically significant.
OM proteome analysis following cold shock in M. catarrhalis
To assess cold shock-induced changes in the OM proteome of M. catarrhalis, 2-DE analysis was used. OMPs were isolated from a culture of M. catarrhalis strain O35E, which was exposed to a 3-hour cold shock at 26°C or to continuous growth at 37°C. A collection of 6 gels (3 of each temperature) resulting from three independent experiments was analyzed. Three OMPs (~75 kDa, pI9; 50 kDa, pI7; and 14 kDa, pI8) were found to be differentially (a greater than twofold change) regulated in response to a 26°C cold shock (Figure 1). Among these proteins, two spots (75 and 15 kDa) were upregulated and one spot (50 kDa) was down-regulated at 26°C (Figure 1A) in comparison with exposure to 37°C (Figure 1B). The 75 kDa spot, which is upregulated at 26°C, was identified by comparing spot pattern of M. catarrhalis O35E wild-type and O35E.tbpB mutant strain as TbpB (Figure 1C), a peripheral OM lipoprotein possessing transferrin-binding properties, indicating that cold shock may increase iron acquisition, which is important for both growth and virulence.
Increased expression of genes involved in iron acquisition of M. catarrhalisinduced by cold shock
Because lactoferrin rather than transferrin is the primary carrier of iron on mucosal surfaces and lactoferrin binding proteins are thought to be important virulence factors in some gram-negative bacteria , we investigated whether cold shock affects the expression of these genes. As shown in Figure 2, cold shock increased the mRNA level of lbpB and lbpA genes in strain O35E after 3 h of incubation at 26°C (Figure 2C). Furthermore, cold shock increased the transcriptional level of lbpA and lbpB of other clinical isolates indicating that this effect is a general characteristic of M. catarrhalis (Figure 2D).
Enhanced binding of transferrin and lactoferrin on the surface of M. catarrhalisinduced by cold shock
Since lactoferrin is an antibacterial protein found in human secretions , it was important to determine its bactericidal effect on M. catarrhalis. No bactericidal effect was observed when M. catarrhalis strain O35E was incubated with human lactoferrin (data not shown).
Because CopB is involved in the ability of M.catarrhalis to acquire iron from human lactoferrin and transferrin, we assessed the expression of this protein following cold shock. Flow cytometry analysis demonstrates that exposure of M.catarrhalis strain O35E to 26°C increases the expression of CopB on the bacterial surface (Figure 4D and 4E).
Cold shock results in upregulation of UspA2 and increases the binding of vitronectin on the surface of M. catarrhalis
Cold shock influences hag expression and binding of human IgD on the surface of M. catarrhalis
In this study, we have analyzed the cold shock-induced changes in the OM proteome of M. catarrhalis and identified TbpB, whose expression was increased more than two-fold after a 26°C cold shock, as a member of the iron acquisition systems that is important for both growth and virulence. Our data demonstrate that the expression of transferrin receptors and transferrin binding on the bacterial surface were also increased when M. catarrhalis was exposed to a 26°C cold shock. Transferrin is predominantly found in serum and in serous exudates. During pronounced inflammation, it is likely that the local tissue damage results in the transsudation of various iron sources, including transferrin, to mucosal surfaces acting as additional iron sources for M. catarrhalis .
We also demonstrate that a 26°C cold shock increased the expression of genes involved in lactoferrin acquisition and enhanced lactoferrin binding on the surface of M. catarrhalis. The overall presence of lactoferrin receptors in M. catarrhalis isolates suggests its important role in colonization or infection . In our previous study we demonstrated that exposure of M. catarrhalis to 26°C increases the release of proinflammatory cytokine IL-8 in pharyngeal epithelial cells likely leading to the increased inflammation . Thus, greater local concentrations of IL-8 would promote enhanced recruitment and influx of neutrophils that release lactoferrin from their secondary granules, which contribute to lactoferrin levels both locally and in the circulation [33, 34]. On the other hand, increased expression of M. catarrhalis lactoferrin binding proteins following cold shock would facilitate the binding and acquisition of iron from lactoferrin to support growth of bacteria in the mucosal environment. It has been shown that supplemental lactoferrin can enhance the virulence of meningococcal infection in mice . In addition to iron acquisition, lactoferrin receptors may provide protection against anti-bacterial cationic peptides (eg, lactoferricin) and reduce complement-mediated killing. The pneumococcal surface protein PspA binds lactoferrin and protects Streptococcus pneumoniae against the antibacterial effect of lactoferricin . The release of LbpB from the cell surface by a NalP protease protects Neisseria meningitidis against bactericidal antibodies . Therefore, increased expression of lactoferrin receptors and enhanced binding of lactoferrin on the surface of bacteria following cold shock might be associated with enhanced protection of M. catarrhalis against anti-bacterial cationic peptides and bactericidal antibodies.
The level of UspA2 protein that afforded serum resistance in the bactericidal activity assay has been shown to correlate with increased binding of vitronectin . Our results indicate that cold shock upregulates the UspA2 protein expression and promotes M. catarrhalis binding to vitronectin. Increased UspA2 protein expression at 26°C was not the result of higher copy number of uspA2 mRNA, indicating that post-transciptional mechanisms are involved in upregulation of this protein after cold shock . Cold shock did not influence the serum resistance of O35E strain indicating that M. catarrhalis strains may need to maintain a certain threshold level of UspA2 protein necessary to evade host defenses. Most seroresistant M. catarrhalis strains express at 37°C sufficient levels of UspA2 to mediate serum resistance . It is conceivable that cold shock would increase UspA2 expression and vitronectin binding in M. catarrhalis strains constitutively expressing low levels of UspA2, leading to the enhanced serum resistance.
The infant population during the first year of life possesses a substantial proportion of IgD in saliva . The interaction between Hag and IgD mediates B cell endocytosis and killing of M. catarrhalis whereas non-IgD-binding bacteria were not taken up by B cells . Furthermore, IgD-stimulated mucosal basophils release antimicrobial factors inhibiting the replication of M. catarrhalis . Here we demonstrate that cold shock at 26°C reduces the mRNA expression level of hag, Hag protein expression and the Hag-mediated binding of human IgD to the surface of M. catarrhalis. Decreased copy numbers of hag at 26°C were also found in other clinical isolates indicating that this effect is a general characteristic of seroresistant M. catarrhalis . Therefore, reduced expression of Hag and decreased binding of IgD on the bacterial surface following cold shock might lead to reduced stimulation of B cells and increased survival by prevention of endocytosis by these cells as well as to decreased stimulation of basophils leading to reduced release of antimicrobal factors. However, the presence of specific IgD against LOS triggered increased recognition of bacteria following cold shock (Figure 6). Consequently, children who lack LOS-specific IgD may be more susceptible to M. catarrhalis infections, particularly after exposure to cold air.
Three OMPs were found to be differentially (a greater than two fold change) regulated in response to a 26°C cold shock (Figure 1), while immunoblot and flow cytometric analysis revealed that several other OMPs are also involved in cold shock response. The lack of some differentially regulated OMPs in the 2-DE pattern might be the result of difficult identification or low abundance. Furthermore, protein spots with a fold change below the indicated threshold were considered by the Image Master 2D program as not relevant.
Thus, cold shock, which occurs when humans breathe cold air , is a physiologic phenomenon during the cold season and entails a range of adaptive events in the residential upper respiratory tract flora that lead to the stimulation of nutrient (e.g., iron)-acquistion, serum resistance and immune evasion potentially resulting in increased bacterial density on the nasopharyngeal surface. Clinical studies in children have demonstrated that the density of M. catarrhalis in the nasopharynx is positively associated with prolonged respiratory tract symptoms and a greater likelihood of purulent otitis media [40, 41]. This study demonstrates that a 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates both CopB and UspA2 expression, the latter leading to improved vitronectin binding on the surface of bacteria. In contrast, cold shock decreases the expression of Hag and reduces the IgD-binding on the surface of M. catarrhalis. These findings indicate that cold air in the human upper respiratory tract induces in M. catarrhalis a complex of adaptive mechanisms that may enable the bacterium to target their host cellular receptors or soluble effectors and consequently to display enhanced growth, colonization and virulence.
A physiologic cold shock as it occurs when humans breathe cold air for prolonged periods of time increases the capacity of M. catarrhalis for iron uptake from human lactoferrin and transferrin, enhances the capacity of M. catarrhalis to bind vitronectin, which neutralizes the lethal effect of human complement, and decreases IgD-binding by hemagglutinin. These data support the notion that M. catarrhalis uses physiologic exposure to cold air to upregulate pivotal survival systems in the human pharynx that may contribute to bacterial virulence. Thus, cold shock may exert adaptive events in at least one member of the residential upper respiratory tract flora of facultative pathogens, which may increase the bacterial density on the respiratory tract mucosal surface (which in turn is associated with an increased likelihood of acute otitis media).
This work was supported by the Swiss National Science Foundation (SNF) grants 3100A0-102246 and 3100A0-116053 (to CA). The authors thank Dr. Eric Hansen, University of Texas Southwestern Medical Center, Dallas, TX, for the kind gift of the monoclonal antibodies mAb10F3 and mAb17C7.
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